| Objective:To induce Graves Disease model of BABL/c mice through the recombinant adenovirus Ad E-GFP-h TSHR289, and to determine the impact of mi R-346 on Tfh cells in GD mice.Methods:(1)Adenovirus backbone plasmid was transformed into competent state E. coli BJ5183 through electrotransformation technology. and the competent state E. coli BJ5183-p was prepared through Ca Cl2 method.(2)Restriction endonuclease digestion was applied to assert target sequence h TSHR289 into p Ad Track-CMV with green fluorescent protein(GFP). Next, p Ad Track-h TSHR289 was transformed into competent state E.coli BJ5183-p. Then, adenovirus backbone plasmid and p Ad Track-h TSHR289 achieved homologous recombination in E. coli BJ5183 through recombinant enzyme activity. And the recombinat adenovirus backbone plasmid(p AEeasy-GFP-h TSHR289)were obtained. Further, Large-Scale preparation of p Ad Easy-GFP-h TSHR289 were obtained through competent state E. coli DH5α, and then transfected it into 293 A cells. Expression of GFP could be detected under fluorescence microscope in two days after transfection, what is more, transfection efficiency could be estimated.(3)Larges amounts of recombinant adenovirus were amplified and the titers of recombinant adenovirus were determined by TCID50. Female BALB/c mouse was immunized by intramuscular injection of adenovirus On the zero week, the third week and the sixth week. On the 10 th week, serum TRAb and T4 level were detected by ELISA, meanwhile, the mice thyroid glands were dissected for pathological examination.(4)Flow cytometry was used to determine the proportion of Tfh cells both in the spleens and neck lymph nodes of the model mice.(5)Mi R-346 was injected into GD mice on 0 d, 4 d, 8 d, 12 d, 16 d, on 18 d, serum TRAb and T4 level were detected by ELISA, and the neck lymph nodes were taken for Tfh cells examination with Flow cytometry, controls are necessary.Results:(1)Through enzyme digestion, transfer, and sequencing, the target sequence h TSHR289 was inserted into the adenovirus backbone plasmid and the recombinant adenovirus was obtained through 293 A cells.(2)The recombinant adenovirus with high titer and high infectivity. TCID50 shows that the titer of Ad E-GFP-h TSHR289 was 3.5×109 pfu/ml and that of Ad E-GFP-CMV was 2.4×109 pfu/ml. ELISA analysis results suggested that compared with the control group mice, adenoviral Ad EGFP-h TSHR289 treatment group 80% mice serum level of TRAb were significantly increased, also T4 level of 60% mice were higher; thyroid pathological section showed that thyroid cells of the experimental mice are mild hyperplasia, and colloid in follicles is reduced.(3)Compared with the control group, the proportion of Tfh cells in the neck lymph nodes of GD mice was significantly increased, while there was no significant proportion change of Tfh cells in the spleen.(4)After the mi R-346 treatment, compared with the control group, the serum levels of TRAb and T4 in GD mice were alleviated. Besides, the proportion of Tfh cells in the neck lymph nodes also reduced.Conclusion:( 1) Recombinant adenovirus backbone plasmid expressing human TSHR289 was obtained through Adeasy system. The recombinant adenovirus were obtained through 293 A cells.(2)GD mice model was successfully induced by recombinant adenovirus.(3)Tfh in the neck lymph nodes of GD mice was significantly increased.(4)Mi R-346 can down the level of autoantibodies in GD mice, which was induced by Ad E-GFP-h TSHR289.(5)Mi R-346 can alleviate the increase of Tfh cells in the neck lymph nodes of GD model. |
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